High inorganic phosphate concentration inhibits osteoclastogenesis by modulating miR-223

Chronic kidney disease-mineral and bone disorder (CKD-MBD) is a common complication of CKD, and uremic toxins have been shown to be instrumental in this process. We have previously shown that miR-223 is increased in smooth muscle cells subjected to the uremic toxin inorganic phosphate (Pi). In the present study we investigated the influence of this miRNA in osteoclastogenesis in order to elucidate its role in the course of CKD-MBD. RT-qPCR demonstrated that high Pi concentration decreased miR-223 expression in differentiated RAW 264.7 cells. Up- and down-regulation of miR-223 was performed using specific pre-miR and anti-miR-223. Differentiation of monocyte/macrophage precursors was assessed by using RAW 264.7 cells and peripheral blood mononuclear cells (PBMC). TRAP activity and bone resorption were used to measure osteoclast activity. Pi induced a marked decrease in osteoclastogenesis in RAW cells and miR-223 levels were concomitantly decreased. Anti-miR-223 treatment inhibited osteoclastogenesis in the same way as Pi. In contrast, overexpression of miR-223 triggered differentiation, as reflected by TRAP activity. We showed that miR-223 affected the expression of its target genes NFIA and RhoB, but also osteoclast marker genes and the Akt signalling pathway, which induces osteoclastogenesis. These results were confirmed by measuring bone resorption activity of human PBMC differentiated into osteoclasts. We thus demonstrate a role of miR-223 in osteoclast differentiation, with rational grounds to use deregulation of this miRNA to selectively increase osteoclast-like activity in calcified vessels of CKD-MBD. This approach could alleviate vascular calcification without altering bone structure.